Surgical patients who cannot tolerate enteral feedings are critically dependent on total parenteral nutrition (TPN). However, TPN is associated with a loss of epithelial barrier function (EBF), which can lead to intestinal dysfunction and the transit of luminal pathogens and toxins into the host. Using a mouse TPN model, our laboratory has demonstrated that the intestinal intraepithelial lymphocytes (IEL) population undergoes a number of significant changes in phenotype and function, including an increase in interferon gamma (IFN-g), decrease in interleukin-10 (IL-10), and increase in epithelial cell (EC)-membrane-bound TNF-a. These cytokine changes may well be key mechanisms leading to the loss of EBF, and appears to be regulated via the PI3K/p-Akt signaling pathway. TPN resulted in a loss in the phosphorylation of Akt (p-Akt), an ubiquination in b-catenin, and loss of EBF. Interestingly, preliminary data also shows that the addition of glutamine to TPN, exogenous epidermal growth factor (EGF) or the removal of pro-inflammatory cytokines can prevent many of these TPN-associated changes. The overlying hypothesis for this proposal is the TPN administration leads to altered cytokine expression with resultant decline in PI3K/p-Akt signaling that results in loss of EBF. To address this, the following specific aims are proposed. 1. To determine the influence of IEL-derived cytokine expression on the alterations in the EC PI3k/p-Akt signaling pathway. This aim will determine the relevance of the presence or absence of pro-inflammatory cytokines within the mucosa to the abundance of p-Akt. Using novel promoters of this signaling pathway, the aim then proposes to determine the relation of p-Akt expression to the maintenance of EC proliferation and EBF. 2. Examine whether the loss of EGF-receptor expression with TPN results in the loss of p-AKT abundance. The aim will also examine whether improved barrier function with glutamine supplemented TPN is dependent on EGF/EGF-R signaling. Finally, this aim will examine if the TPN- associated decline in EGF signaling can be prevented by manipulation of IEL-derived cytokine expression. 3. Determine how alteration in TNF-a receptor abundance during TPN administration alters EBF. This aim will examine changes in the abundance of TNF-a and its receptors in the formation of TPN-associated decline in EBF. As EGF signaling can be dependent on TNF-a, the interdependence of these factors will also be examined. Finally, a disintegrin and metalloproteinase 17 (ADAM17) can shed the active ligands for EGF-R, and TNF-a receptors. The relation of ADAM17 and the loss of EBF will be explored. 4. This aim will measure changes in intestinal mucosal immunocyte phenotype and function during pediatric and adult administration of TPN, and correlate these with associated loss of epithelial barrier function. These experiments will elucidate many of the mechanisms driving loss of EBF associated with TPN. Beyond this goal, the information derived from this proposal will augment our basic understanding of lymphoid / EC interactions, which may have relevance to a number of other gastrointestinal disease processes. PUBLIC HEALTH RELEVANCE: Surgical patients who cannot tolerate enteral feedings are critically dependent on total parenteral nutrition (TPN), or intravenous nutrition. However, TPN is associated with a loss of epithelial barrier function (EBF), which can lead to the transit of luminal pathogens and toxins into the host. Using a mouse model of TPN and novel transgenic mice and promoters and inhibitors of intracellular signaling this proposal will examine the mechanisms which lead to this loss of EBF. Beyond this goal, the information derived from this proposal will augment our basic understanding of a number of other gastrointestinal disease processes where barrier function is lost, including inflammatory bowel disease and infectious disorders of the intestine.